Binding of herpes simplex virus-1 US11 to specific RNA sequences.
Herpes simplex virus-1 US11 is a RNA-binding protein with a novel RNA-binding domain. US11 has been reported to exhibit sequence- and conformation-specific RNA-binding, but the sequences and conformations important for binding are not known. US11 has also been described as a double-stranded RNA (dsRNA)-binding protein. To investigate the US11-RNA interaction, we performed in vitro selection of RNA aptamers that bind US11 from a RNA library consisting of >10(14) 80 base sequences which differ in a 30 base randomized region. US11 bound specifically to selected aptamers with an affinity of 70 nM. Analysis of 23 selected sequences revealed a strong consensus sequence. The US11 RNA-binding domain and < or =46 bases of selected RNA containing the consensus sequence were each sufficient for binding. US11 binding protected the consensus motif from hydroxyl radical cleavage. RNase digestions of a selected aptamer revealed regions of both single-stranded RNA and dsRNA. We observed that US11 bound two different dsRNAs in a sequence non-specific manner, but with lower affinity than it bound selected aptamers. The results define a relatively short specific sequence that binds US11 with high affinity and indicate that dsRNA alone does not confer high-affinity binding.
Bryant
Kevin F
KF
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Avenue, Boston, MA 02115, USA.
Cox
J Colin
JC
Wang
Hongming
H
Hogle
James M
JM
Ellington
Andrew D
AD
Coen
Donald M
DM
D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D024202:Electrophoretic Mobility Shift Assay; D018259:Herpesvirus 1, Human; D017665:Hydroxyl Radical / Q000737:chemistry; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*
D009841:Oligonucleotides; D012330:RNA, Double-Stranded; D016601:RNA-Binding Proteins; C074546:US11 protein, herpesvirus; D014764:Viral Proteins; D017665:Hydroxyl Radical; D012313:RNA; D012260:Ribonucleases