Sindbis virus RNA polymerase is degraded by the N-end rule pathway.

Abstract

Upon infection of animal cells by Sindbis virus, four nonstructural (ns) proteins, termed nsPl-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 2 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 requlres readthrough of an opal termination codon such that it is underproduced; 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.Sindbis virus, the prototype alphavirus, is an RNA animal virus whose genome is 11,703 nucleotides long. Four nonstructural (ns) proteins, called nsPl-4 from their order in the genome, are required for replication and transcription of viral RNAs and are translated as polyprotein precursors from the genomic RNA (1). We have previously reported that the synthesis of nsP4, which is thought to be the viral RNA polymerase (2-4), is regulated by at least two mechanisms in Sindbis virus. First, nsP4 is produced only upon read-through of an opal codon located at the 3' end of the nsP3 gene (5). As a consequence, nsP4 and nsP4-containing polyproteins are underproduced compared with nsP1, -2, and -3. The second regulatory mechanism involves differential processing of Sindbis polyprotein precursors by a set of virusencoded proteinases whose active site is located in the C-terminal half of nsP2 (6, 7). Polyprotein precursors containing nsP2 are all proteolytically active but differ in their cleavage-site specificities: those containing nsP1 are unable to cleave between nsP2 and nsP3, whereas only those containing nsP3 cleave between nsP3 and nsP4 (8, 9). Apparently, these differences lead to a temporal regulation of processing such that very early in infection (0-2 hr after infection) nsP4 is produced (9), whereas later in infection polyprotein P34 is produced rather than nsP4 (9-11). We have proposed that this temporal regulation ofthe relative am...

Systematic, genome-wide identification of host genes affecting replication of a positive-strand RNA virus

“…6 and references therein). The 2a-homologous nsP4 polymerase of Sindbis virus, another member of the alphavirus superfamily, is regulated by the ubiquitin͞proteasome-dependent N-end rule degradation pathway (40). Absence of several other yeast genes involved in ubiquitination or protein turnover also dramatically reduced BMV-directed Rluc expression (Table 1).…”

Regulation of the Sequential Processing of Semliki Forest Virus Replicase Polyprotein

“…P34 is cleaved by free nsP2 resulting in the aggregation of nsP3 in the cytoplasm (18), whereas nsP4 is rapidly degraded by the ubiquitin pathway ( Fig. 8B) (20,39).…”

The N‐end rule pathway of protein degradation

“…The nsP4 protein bears N-terminal Tyr (a primary destabilizing residue; Figs 1 and 5A), and is degraded by the N-end rule pathway in reticulocyte extract (deGroot et al 1991). Tyr is an N-terminal residue of other alphaviral RNA polymerases as well (Strauss & Strauss 1994), suggesting that these homologues of Sindbis polymerase are also degraded by the N-end rule pathway.…”