Phosphorylation of the avian retrovirus integration protein and proteolytic processing of its carboxyl terminus.
The integration protein (IN) of the Prague A strain of Rous sarcoma virus (RSV) was analyzed by high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three polypeptides of similar proportions and molecular mass (32 kDa) were immunoprecipitated by an antiserum directed against the first 10 amino acids of the amino terminus of IN. However, the faster-migrating nonphosphorylated polypeptide was not immunoprecipitated by two different polyclonal antisera directed against the last 11 amino acids of the carboxyl terminus of IN. These results suggest that the faster-migrating species was proteolytically processed at its carboxyl terminus. RSV IN is phosphorylated on an S residue located five amino acids from its carboxyl terminus. Two different missense mutations at this S residue resulted in the isolation of slow-growing viable mutants whose phenotypes were stable. Each mutation at residue 282 eliminated both major phosphorylated-Ser-containing tryptic peptides observed with wild-type IN. An S----F mutation resulted in the conversion of all IN polypeptides to one species that was not precipitable by carboxyl-terminal antisera, suggesting that this amino acid transition promoted proteolysis at the carboxyl terminus. An S----D mutation resulted in the recovery of one major (greater than 95%) slower-migrating polypeptide that was immunoprecipitated by carboxyl-terminal antisera, suggesting that this negatively charged D residue (similar to phosphorylated Ser) inhibited proteolysis. Modification of the S residue at amino acid 262 to R had no apparent effect on the proteolytic processing or phosphorylation of IN.
Horton
R
R
St. Louis University Medical Center, Institute for Molecular Virology, Missouri 63110.
Mumm
S R
SR
Grandgenett
D P
DP
D000595:Amino Acid Sequence; D000818:Animals; D001358:Avian Sarcoma Viruses / Q000201:enzymology* / Q000235:genetics; D002478:Cells, Cultured; D002642:Chick Embryo; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019426:Integrases; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010449:Peptide Mapping; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D014162:Transfection
D015345:Oligonucleotide Probes; D004254:DNA Nucleotidyltransferases; D019426:Integrases