Perturbation of the carboxy terminus of HIV-1 Rev affects multimerization on the Rev responsive element.
Perturbations within the transactivation and carboxy-terminal domains of HIV-1 Rev were examined for effects on Rev responsive element (RRE) binding activities in vitro and biological activity in vivo. Binding affinities, specificities, and multimerization of the transactivation mutants M10 and Rev/Rex M10-16 on the RRE were equivalent to wild-type Rev. Substitution of the Rex transactivation domain within Rev resulted in the incorporation of an internal methionine residue which, when cleaved with CNBr and subsequently purified, produced a protein species (CNBr-Rev) unable to fully multimerize on the RRE. Instead, two discrete protein-dependent species were generated in the gel shift assay. Furthermore, CNBr-Rev was observed to bind to the RRE with high specificity and an equilibrium binding constant of 6 x 10(-10) M. A C-terminal Rev deletion mutant (Rev M9 delta 14) lacking amino acids 68-112 displayed identical RRE binding characteristics to the CNBr-Rev protein. This protein, which lacks both the activation and the C-terminal domains, was biologically inactive but maintained the ability to discriminate the RRE from nonspecific RNA. Deletion of amino acids 92-112 resulted in a Rev mutant (Rev M11 delta 14) which bound to the RRE with wild-type affinity and high specificity. This purified mutant was observed to be aberrant in multimerization activity on the RRE with reduced multimerization apparent in the gel shift assay. However, Rev M11 delta 14 possessed biological activity equivalent to wild-type Rev in a cell-based p24 ELISA assay. These results suggest that polymerization on the RRE is dispensable for Rev activity and that two monomeric Rev proteins bound to the RRE are sufficient for biological activity. Furthermore, in vivo experiments using the Rev/Rex chimeric mutant and the M10 transdominant mutant as well as in vitro dissociation rate studies with Rev M11 delta 14 and Rev M9 delta 14 suggest that the M9 through M11 domain of the protein may be involved in RRE-dependent specific Rev dimerization.
Daly
T J
TJ
Repligen Corporation, Cambridge, Massachusetts 02139.
Rennert
P
P
Lynch
P
P
Barry
J K
JK
Dundas
M
M
Rusche
J R
JR
Doten
R C
RC
Auer
M
M
Farrington
G K
GK
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D003488:Cyanogen Bromide; D004279:DNA, Viral; D015694:Gene Products, rev / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000737:chemistry*; D008969:Molecular Sequence Data; D009154:Mutation; D012367:RNA, Viral / Q000378:metabolism*; D017384:Sequence Deletion; D015533:Transcriptional Activation; D014162:Transfection; D054321:rev Gene Products, Human Immunodeficiency Virus
D004279:DNA, Viral; D015694:Gene Products, rev; D012367:RNA, Viral; D054321:rev Gene Products, Human Immunodeficiency Virus; D003488:Cyanogen Bromide