Requirement for HIV-1 TAR sequences for Tat activation in rodent cells.
HIV-1 gene expression is activated via an interaction between the virally encoded Tat protein and a target RNA, TAR. TAR is located at the immediate 5' end of all viral mRNAs and comprises a partially base-paired stem with a tripyrimidine bulge in the upper stem and a hexanucleotide loop. In vitro, Tat binds specifically to the bulge and upper stem region with no requirement for the loop. In contrast, when Tat activation is analyzed in primate cells, mutations in the loop abolish activation, suggesting a critical role for loop binding cellular factors. However, in rodent cells the reverse is true. Messages with a mutation in the TAR loop are activated whereas messages harboring a wild-type TAR sequence are not activated. By testing the effect of mutations in the bulge and stem in the context of mutation in the loop we now show that this loop-independent activation by Tat in rodent cells requires the critical bulge-stem sequences needed for Tat binding in vitro. These data suggest that in rodent cells, as in vitro, Tat does not require a loop binding cofactor. In rodent cells containing human chromosome 12 (CHO12), however, Tat activation is both bulge and loop dependent. It appears that rodent cells, but not CHO12 cells, are refractory to the normal Tat/TAR activation pathway not by virtue of lacking a loop binding cofactor, but rather by the presence of a loop binding inhibitor of either Tat binding or the activation process.
Sutton
J A
JA
Department of Biochemistry, University of Oxford.
Braddock
M
M
Kingsman
A J
AJ
Kingsman
S M
SM
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D002881:Chromosomes, Human, Pair 12; D006224:Cricetinae; D015696:Gene Products, tat / Q000378:metabolism*; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009843:Oligoribonucleotides; D012313:RNA / Q000235:genetics / Q000378:metabolism; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D009843:Oligoribonucleotides; D054322:tat Gene Products, Human Immunodeficiency Virus; D012313:RNA; D015500:Chloramphenicol O-Acetyltransferase