Functional characterization of Kaposi's sarcoma-associated herpesvirus ORF45 by bacterial artificial chromosome-based mutagenesis

J Virol. 2006 Dec;80(24):12187-96. doi: 10.1128/JVI.01275-06. Epub 2006 Oct 11.

Abstract

Open reading frame 45 (ORF45) of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes an immediate-early protein. This protein is also present in virions as a tegument protein. ORF45 protein interacts with interferon regulatory factor 7 (IRF-7) and inhibits virus-induced type I interferon production by blocking activation of IRF-7. To define further the function of ORF45 and the mechanism underlying its action, we constructed an ORF45-null recombinant virus genome (BAC-stop45) by using a bacterial artificial chromosome (BAC) system. Stable 293T cells carrying the BAC36 (wild type) and BAC-stop45 genomes were generated. When monolayers of 293T BAC36 and 293T BAC-stop45 cells were induced with 12-O-tetradecanoylphorbol-13-acetate and sodium butyrate, no significant difference was found between them in overall viral gene expression and lytic DNA replication, but induced 293T BAC-stop45 cells released 10-fold fewer virions to the medium than did 293T BAC36 cells. When ORF45-null virus was used to infect cells, lower infectivity was observed than for wild-type BAC36. These results suggest that KSHV ORF45 plays roles in both early and late stages of viral infection, probably in viral ingress and egress.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural

MeSH terms

  • Blotting, Western
  • Cell Line
  • Chromosomes, Artificial, Bacterial
  • DNA Primers
  • Escherichia coli
  • Gene Components
  • Gene Expression*
  • Herpesvirus 8, Human / genetics*
  • Humans
  • Immediate-Early Proteins / genetics*
  • Immediate-Early Proteins / metabolism
  • Mutagenesis
  • Reverse Transcriptase Polymerase Chain Reaction
  • Viral Proteins / genetics*
  • Viral Proteins / metabolism
  • Virion / genetics
  • Virion / physiology*

Substances

  • DNA Primers
  • Immediate-Early Proteins
  • ORF45 protein, Kaposi's sarcoma-associated herpesvirus
  • Viral Proteins