Isolation and characterization of an oligomerization-negative mutant of HIV-1 integrase.
The yeast two-hybrid method was used to screen mutagenized DNAs to isolate a variant of the human immunodeficiency virus type 1 integrase (IN) that does not interact with the wild-type IN. The responsible mutation, leading to a single amino acid change (V260E) in the C-terminal domain of IN, blocks IN-IN multimerization but has only small effect on binding to a host interacting protein, INI1 (hSNF5). Binding studies in vitro confirmed the defect in multimerization of the mutant IN. Biochemical analyses of the mutant IN enzyme expressed in bacteria detected only subtle changes in its properties, suggesting that the yeast system is a sensitive reporter of correct IN conformation. Mutant virus carrying the V260E substitution was blocked in replication at the time of DNA integration, consistent with IN multimerization being important for its activity in vivo.
Kalpana
G V
GV
Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx, New York, 10461, USA.
Reicin
A
A
Cheng
G S
GS
Sorin
M
M
Paik
S
S
Goff
S P
SP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001704:Biopolymers; D015153:Blotting, Western; D019556:COS Cells; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins / Q000378:metabolism; D019427:HIV Integrase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D000071796:SMARCB1 Protein; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017422:Sequence Analysis, DNA; D014157:Transcription Factors; D016662:Virus Integration; D014779:Virus Replication
D001704:Biopolymers; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D000071796:SMARCB1 Protein; C513266:SMARCB1 protein, human; C066190:SNF5 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D019427:HIV Integrase