Radio-sequence analysis of in Vivo multilabeled nonstructural protein ns86 of semliki forest virus

Abstract

The N-terminal structure of in vivo multilabeled virus-specific enzymes and other proteins can be determined by single radio-sequence analyses. We now show this for Semliki Forest virus nonstructural protein ns86. The viral proteins were labeled with mixtures of up to seven different tritiated amino acids in cultures of chick embryo cells infected with the is-1 mutant. The multilabeled ns86 was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently degraded together with a carrier protein in a liquid phase sequencer. The resulting amino acid derivatives from each cycle of degradation were then separated by high-performance liquid chromatography and the radioactivity of each fraction was determined. The sequence obtained is sufficient to localize the nucleotide sequence on the 42 S RNA genome that codes for the N-terminus of ns86. Considerable differences were found in the incorporation of the labeled amino acids into ns86 during the in vivo conditions used. Stabilities and chromatographic separations of the amino acid derivatives vary and require extra precautions in radio-sequencing of multilabeled proteins.