Sindbis Virus RNA Polymerase is Degraded by the N-End Rule Pathway

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 88; no. 20; pp. 8967 - 8971
• Main Authors: de Groot, Raoul J., Rumenapf, Tillmann, Kuhn, Richard J., Strauss, Ellen G., Strauss, James H.
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 15.10.1991; National Acad Sciences
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Biological and medical sciences; Dipeptides - pharmacology; DNA-Directed RNA Polymerases - genetics; DNA-Directed RNA Polymerases - metabolism; Fundamental and applied biological sciences. Psychology; Genome, Viral; Genomes; Half lives; Kinetics; Microbiology; Molecular Sequence Data; Mutagenesis, Insertional; Nucleotides; Oligonucleotides; Plasmids; Polymerase Chain Reaction; Polyproteins; Protein Processing, Post-Translational; Proteins; Rabbits; Replicative cycle, interference, host-virus relations, pathogenicity, miscellaneous strains; Restriction Mapping; Reticulocytes; Reticulocytes - metabolism; RNA; Sindbis Virus; Sindbis Virus - enzymology; Sindbis Virus - genetics; Virology; Viruses; Ubiquitin; Lysate; Stability; Enzyme; Site directed mutagenesis; Togaviridae; Rabbit; RNA polymerase; Lagomorpha; Sindbis virus; Proteins; Virus; Vertebrata; Mammalia; Enzymatic digestion; Alphavirus; Reticulocyte; Mechanism of action; N terminal aminoacid; Cell free system
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.88.20.8967

Upon infection of animal cells by Sindbis virus, four nonstructural (ns) proteins, termed nsP1-4 in order from 5' to 3' in the genome, are produced by posttranslational cleavage of a polyprotein. nsP4 is believed to function as the viral RNA polymerase and is short-lived in infected cells. We show here that nsP4 produced in reticulocyte lysates is degraded by the N-end rule pathway, one ubiquitin-dependent proteolytic pathway. When the N-terminal residue of nsP4 is changed by mutagenesis, the metabolic stabilities of the mutant nsP4s follow the N-end rule, in that the half-life of nsP4 bearing different N-terminal residues decreases in the order Met > Ala > Tyr ≥ Phe > Arg. Addition of dipeptides Tyr-Ala, Trp-Ala, or Phe-Ala to the translation mixture inhibits degradation of Tyr-nsP4 and Phe-nsP4, but not of Arg-nsP4. Conversely, dipeptides His-Ala, Arg-Ala, and Lys-Ala inhibit the degradation of Arg-nsP4 but not of Tyr-nsP4 or Phe-nsP4. We found that there is no lysine in the first 43 residues of nsP4 that is required for its degradation, indicating that a more distal lysine functions as the ubiquitin acceptor. Strict control of nsP4 concentration appears to be an important aspect of the virus life cycle, since the concentration of nsP4 in infected cells is regulated at three levels: translation of nsP4 requires readthrough of an opal termination codon such that it is under-produced; differential processing by the virus-encoded proteinase results in temporal regulation of nsP4; and nsP4 itself is a short-lived protein degraded by the ubiquitin-dependent N-end rule pathway.