Functional characterization of Kaposi's sarcoma-associated herpesvirus ORF45 by bacterial artificial chromosome-based mutagenesis.
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.
Zhu
Fan Xiu
FX
Department of Microbiology, School of Dental Medicine, University of Pennsylvania, 240 S. 40th Street, Philadelphia, PA 19104, USA.
Li
Xiaojuan
X
Zhou
Fuchun
F
Gao
Shou-Jiang
SJ
Yuan
Yan
Y
D015153:Blotting, Western; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial; D017931:DNA Primers; D004926:Escherichia coli; D040461:Gene Components; D015870:Gene Expression; D019288:Herpesvirus 8, Human / Q000235:genetics*; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics* / Q000378:metabolism; D016296:Mutagenesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D014771:Virion / Q000235:genetics / Q000502:physiology*
D017931:DNA Primers; D017874:Immediate-Early Proteins; C456286:ORF45 protein, Kaposi's sarcoma-associated herpesvirus; D014764:Viral Proteins