Exploring the DNA binding interactions of the Kaposi's sarcoma-associated herpesvirus lytic switch protein by selective amplification of bound sequences in vitro.
The lytic switch protein RTA of Kaposi's sarcoma-associated herpesvirus (KSHV) can be targeted to DNA by either direct sequence-specific recognition or via protein-protein interactions with host transcription factors. We have searched for sequences capable of direct RTA binding by screening synthetic oligonucleotide pools and KSHV genomic libraries for RTA-interacting elements, using repeated cycles of in vitro binding followed by amplification of the bound sequences. Multiple low-affinity sequences were recovered from the random pools, with generation of only a weak consensus sequence. The genomic library, by contrast, yielded many biologically relevant fragments, most of which could be shown to interact with RTA in vitro and some of which likely play important regulatory roles in vivo. Surprisingly, the most highly selected fragment came from the promoter of a late gene (gB) and contained at least two direct RTA binding sites, as well as one RBP-Jkappa binding site. This raises the possibility that some late KSHV genes may also be subject to direct RTA regulation, though indirect models are not excluded.
Ziegelbauer
Joseph
J
G.W. Hooper Foundation, University of California, San Francisco, Box 0552, 513 Parnassus Ave., San Francisco, California 94143-0552, USA.
Grundhoff
Adam
A
Ganem
Don
D
D001483:Base Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D019288:Herpesvirus 8, Human / Q000235:genetics / Q000378:metabolism*; D017874:Immediate-Early Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D020411:Oligonucleotide Array Sequence Analysis; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; D009841:Oligonucleotides; C417604:Rta protein, Human herpesvirus 8; D015534:Trans-Activators; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C116207:glycoprotein B, human herpesvirus 8