Extensive sequence-specific information throughout the CAR/RRE, the target sequence of the human immunodeficiency virus type 1 Rev protein.
The significance and location of sequence-specific information in the CAR/RRE, the target sequence for the Rev protein of the human immunodeficiency virus type 1 (HIV-1), have been controversial. We present here a comprehensive experimental and computational approach combining mutational analysis, phylogenetic comparison, and thermodynamic structure calculations with a systematic strategy for distinguishing sequence-specific information from secondary structural information. A target sequence analog was designed to have a secondary structure identical to that of the wild type but a sequence that differs from that of the wild type at every position. This analog was inactive. By exchanging fragments between the wild-type sequence and the inactive analog, we were able to detect an unexpectedly extensive distribution of sequence specificity throughout the CAR/RRE. The analysis enabled us to identify a critically important sequence-specific region, region IIb in the Rev-binding domain, strongly supports a proposed base-pairing interaction in this location, and places forceful constraints on mechanisms of Rev action. The generalized approach presented can be applied to other systems.
Dayton
E T
ET
Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892.
Konings
D A
DA
Powell
D M
DM
Shapiro
B A
BA
Butini
L
L
Maizel
J V
JV
Dayton
A I
AI
D001483:Base Sequence; D015694:Gene Products, rev / Q000235:genetics*; D015750:Genes, gag; D015757:Genes, rev; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology; D008957:Models, Genetic; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D010802:Phylogeny; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication / Q000235:genetics; D054321:rev Gene Products, Human Immunodeficiency Virus
D015694:Gene Products, rev; D012367:RNA, Viral; D054321:rev Gene Products, Human Immunodeficiency Virus