title: Kaposi's sarcoma-associated herpesvirus ORF18 and ORF30 are essential for late gene expression during lytic replication.
abstract: Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with several human malignances. As saliva is likely the major vehicle for KSHV transmission, we studied in vitro KSHV infection of oral epithelial cells. Through infection of two types of oral epithelial cells, normal human oral keratinocytes (NHOKs) and papilloma-immortalized human oral keratinocyte (HOK16B) cells, we found that KSHV can undergo robust lytic replication in oral epithelial cells. By employing de novo lytic infection of HOK16B cells, we studied the functions of two previously uncharacterized genes, ORF18 and ORF30, during the KSHV lytic cycle. For this purpose, an ORF18-deficient virus and an ORF30-deficient virus were generated using a mutagenesis strategy based on bacterial artificial chromosome (BAC) technology. We found that neither ORF18 nor ORF30 is required for immediately early or early gene expression or viral DNA replication, but each is essential for late gene expression during both de novo lytic replication and reactivation. This critical role of ORF18 and ORF30 in late gene expression was also observed during KSHV reactivation. In addition, global analysis of viral transcripts by RNA sequencing indicated that ORF18 and ORF30 control the same set of viral genes. Therefore, we suggest that these two viral ORFs are involved in the same mechanism or pathway that coregulates the viral late genes as a group.
  
  
  OBJECTIVE
  While KSHV can infect multiple cell types in vitro, only a few can support a full lytic replication cycle with progeny virions produced. Consequently, KSHV lytic replication is mostly studied through reactivation, which requires chemicals to induce the lytic cycle or overexpression of the viral transcriptional activator, RTA. In this study, we present a robust de novo lytic infection system based on oral epithelial cells. Using this system, we demonstrate the role of two viral ORFs, ORF18 and ORF30, in regulating viral gene expression during KSHV lytic replication. As the major route of KSHV transmission is thought to be via saliva, this new KSHV lytic replication system will have important utility in the field.
authors: Department of Molecular and Medical Pharmacology, University of California, Los Angeles, California, USA.
authors: Danyang
authors: D
authors: Gong
authors: Department of Molecular and Medical Pharmacology, University of California, Los Angeles, California, USA.
authors: Nicholas C
authors: NC
authors: Wu
authors: Department of Molecular and Medical Pharmacology, University of California, Los Angeles, California, USA.
authors: Yafang
authors: Y
authors: Xie
authors: Department of Molecular and Medical Pharmacology, University of California, Los Angeles, California, USA.
authors: Jun
authors: J
authors: Feng
authors: Department of Molecular and Medical Pharmacology, University of California, Los Angeles, California, USA.
authors: Leming
authors: L
authors: Tong
authors: Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.
authors: Kevin F
authors: KF
authors: Brulois
authors: Department of Molecular and Medical Pharmacology, University of California, Los Angeles, California, USA.
authors: Harding
authors: H
authors: Luan
authors: Department of Molecular and Medical Pharmacology, University of California, Los Angeles, California, USA.
authors: Yushen
authors: Y
authors: Du
authors: Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.
authors: Jae U
authors: JU
authors: Jung
authors: School of Dentistry, University of California, Los Angeles, California, USA.
authors: Cun-yu
authors: CY
authors: Wang
authors: School of Dentistry, University of California, Los Angeles, California, USA.
authors: Mo Kwan
authors: MK
authors: Kang
authors: School of Dentistry, University of California, Los Angeles, California, USA Department of Medicine, University of California, Los Angeles, California, USA.
authors: No-Hee
authors: NH
authors: Park
authors: Department of Molecular and Medical Pharmacology, University of California, Los Angeles, California, USA rsun@mednet.ucla.edu twu@mednet.ucla.edu.
authors: Ren
authors: R
authors: Sun
authors: Department of Molecular and Medical Pharmacology, University of California, Los Angeles, California, USA rsun@mednet.ucla.edu twu@mednet.ucla.edu.
authors: Ting-Ting
authors: TT
authors: Wu
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial; D015967:Gene Expression Regulation, Viral; D019288:Herpesvirus 8, Human / Q000235:genetics* / Q000378:metabolism; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D015603:Keratinocytes / Q000473:pathology / Q000821:virology*; D008969:Molecular Sequence Data; D009061:Mouth Mucosa / Q000473:pathology / Q000821:virology; D016366:Open Reading Frames; D017384:Sequence Deletion; D015398:Signal Transduction; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D014775:Virus Activation; D014779:Virus Replication
chemical_list: D014764:Viral Proteins