Inhibition of transcription by the TAR RNA of HIV-1 in a nuclear extract of HeLa cells.
Regulation of transcription of human immunodeficiency virus type-1 (HIV-1) requires specific interaction of Tat protein with the trans-activation response region (TAR). Inhibition of replication of HIV-1 has previously been achieved with a TAR decoy, namely a short RNA oligonucleotide that corresponded to the sequence of the authentic TAR RNA. Since TAR RNA has the potential to interact with cellular factors, we examined the effect of TAR RNA on efficiency of transcription in nuclear of HeLa cell extracts. We performed an in vitro transcription assay in the presence of authentic TAR RNA using a template that was driven by the CMV (cytomegalovirus) early promoter in a HeLa nuclear extract and found, for the first time, that TAR RNA inhibited transcription by approximately 60-70% independently of the Tat-TAR interaction. Furthermore, we evaluated inhibition of transcription by variants of TAR RNA and found that the TAR RNA loop, bases surrounding the loop, the triple base bulge and the 'lower' stem region of TAR RNA were responsible for the inhibition of transcription. Taken together, earlier reports on proteins that bind to TAR RNA and the present results suggest that integrity of TAR RNA is important for efficient binding to cellular transcription factors. As judged from the significant inhibition observed in this study, the TAR decoy might sequester transcription factors and thus it might potentially be able to inhibit transcription of housekeeping genes that are unrelated to Tat function.
Yamamoto
R
R
National Institute of Bioscience and Human Technology, AIST, MITI, Tsukuba Science City, Ibaraki 305, Japan.
Koseki
S
S
Ohkawa
J
J
Murakami
K
K
Nishikawa
S
S
Taira
K
K
Kumar
P K
PK
D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral; D015696:Gene Products, tat / Q000378:metabolism / Q000494:pharmacology*; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology; D006367:HeLa Cells / Q000648:ultrastructure*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic; D014779:Virus Replication / Q000187:drug effects; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D012367:RNA, Viral; D054322:tat Gene Products, Human Immunodeficiency Virus