Analysis of mutations in the integration function of Moloney murine leukemia virus: effects on DNA binding and cutting.
The 3' terminus of the pol gene of Moloney murine leukemia virus encodes the integration (IN) protein, required for the establishment of the integrated provirus. A series of six linker insertion mutations and two single-base substitutions were generated within the region encoding the IN protein. Mutations were initially generated within an Escherichia coli plasmid expressing the IN protein, and the resulting variants were assayed for DNA-binding activity. Mutations which altered conserved cysteine residues within a potential DNA finger-binding motif resulted in lower or variable DNA binding, which appeared to be the result of variable protein folding. Upon renaturation, these proteins were able to nonspecifically bind DNA in a manner similar to that of the other mutant IN proteins and the parent. When reconstructed back into full-length virus, seven of the eight mutations were lethal. All mutants produced a stable IN protein in virions and mediated normal conversion of the retroviral RNA to its three DNA forms. Fine-structure analysis of the linear double-stranded viral DNA indicated that all seven lethal alterations within the IN protein blocked the formation of the 3' recessed termini that normally precedes integration.
Department of Biochemistry, University of Medicine and Dentistry of New Jersey/Robert Wood Johnson Medical School, Piscataway 08854.
M J
MJ
Roth
P
P
Schwartzberg
N
N
Tanese
S P
SP
Goff
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D002472:Cell Transformation, Viral; D004254:DNA Nucleotidyltransferases / Q000235:genetics; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000235:genetics; D015751:Genes, pol; D019426:Integrases; D008242:Lysogeny; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000235:genetics* / Q000502:physiology; D009154:Mutation; D015183:Restriction Mapping
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D004254:DNA Nucleotidyltransferases; D019426:Integrases