The termination of protein synthesis in ribosomes is governed by termination (stop) codons in messenger RNAs and by polypeptide chain release factors (RFs). Although the primary structure of prokaryotic RFs and yeast mitochrondrial RF is established, that of the only known eukaryotic RF (eRF) remains obscure. Here we report the assignment of a family of tightly related proteins (designated eRF1) from lower and higher eukaryotes which are structurally and functionally similar to rabbit eRF. Two of these proteins, one from human and the other from Xenopus laevis, have been expressed in yeast and Escherichia coli, respectively, purified and shown to be active in the in vitro RF assay. The other protein of this family, sup45 (sup1) of Saccharomyces cerevisiae, is involved in omnipotent suppression during translation. The amino-acid sequence of the eRF1 family is highly conserved. We conclude that the eRF1 proteins are directly implicated in the termination of translation in eukaryotes.

Turnip yellow mosaic virus (TYMV) encodes a 206-kDa (206K) polyprotein with domains of methyltransferase, proteinase, NTPase/helicase, and RNA-dependent RNA polymerase (RdRp). In vitro, the 206K protein has been shown to undergo proteolytic processing, giving rise to the synthesis of 140-kDa (140K) and 66-kDa (66K) proteins, the latter comprising the RdRp protein domain. Antibodies were raised against the 66K protein and were used to detect the corresponding viral protein in infected cells; both leaf tissues and protoplasts were examined. The antiserum specifically recognized a protein of approximately 66 kDa, indicating that the cleavage observed in vitro is also functional in vivo. The 66K protein accumulates transiently during protoplast infection and localizes to cellular membrane fractions. Indirect immunofluorescence assays and electron microscopy of immunogold-decorated ultrathin sections of infected leaf tissue using anti-66K-specific antibody revealed labeling of membrane vesicles located at the chloroplast envelope.

. During viral infection, the 66K protein localizes to virus-induced chloroplastic membrane vesicles, which are closely associated with TYMV RNA replication. To investigate the determinants of its subcellular localization, the 66K protein was expressed in plant protoplasts from separate plasmids. Green fluorescent protein (GFP) fusion and immunofluorescence experiments demonstrated that the 66K protein displayed a cytoplasmic distribution when expressed individually but that it was relocated to the chloroplast periphery under conditions in which viral replication occurred. The 66K protein produced from an expression vector was functional in viral replication since it could transcomplement a defective replication template. Targeting of the 66K protein to the chloroplast envelope in the course of the viral infection appeared to be solely dependent on the expression of the 140K protein. Analysis of the subcellular localization of the 140K protein fused to GFP demonstrated that it is targeted to the chloroplast envelope in the absence of other viral factors and that it induces the clumping of the chloroplasts, one of the typical cytological effects of TYMV infection. These results suggests that the 140K protein is a key organizer of the assembly of the TYMV replication complexes and a major determinant for their chloroplastic localization and retention.A universal feature of eukaryotic positive-strand RNA viruses is that replication of their genomes is closely associated with intracellular membranes (reviewed in reference 8). Most purified viral RNA replication complexes copurify with membrane extracts from infected cells (reviewed in reference 10) and, although in some cases RNA synthesis activity can be solubilized (24, 67), in vivo and in vitro studies suggest that the presence of membranes and/or phospholipids is essential for at least some steps of RNA replication (37,41,67). It was proposed that these membranes can play both a structural and a functional role in the replication complex.Electron microscopy observations of infected cells revealed that many positive-stranded RNA viruses induce proliferation and/or reorganization of the intracellular membranes of their host to create a membrane compartment in which RNA replication takes place. Depending on the virus, a variety of membrane systems can be concerned, including the early and late endomembrane systems (52, 59), the nuclear envelope (13), the vacuole (64), the endosomes and lysosomes (17, 59), the peroxisomes (56), chloroplasts (35), and mitochondria (14, 40). The fact that distinct types of membranes are involved in the replication of different viruses suggests the establishment of specific interactions between such host membranes and virusencoded proteins. A number of viral proteins that target replication complexes to intracellular membranes have been identified (9,48,55,63). Membrane interaction of host-encoded factors that are part of the viral replication complex has also been reported (22,68).Despite this universal association of positive-strand ...

Replication of positive-strand RNA viruses, the largest group of plant viruses, is initiated by viral RNA-dependent RNA polymerase (RdRp). Given its essential function in viral replication, understanding the regulation of RdRp is of great importance. Here, we show that Turnip yellow mosaic virus (TYMV) RdRp (termed 66K) is degraded by the proteasome at late time points during viral infection and that the accumulation level of 66K affects viral RNA replication in infected Arabidopsis thaliana cells. We mapped the cis-determinants responsible for 66K degradation within its N-terminal noncatalytic domain, but we conclude that 66K is not a natural N-end rule substrate. Instead, we show that a proposed PEST sequence within 66K functions as a transferable degradation motif. In addition, several Lys residues that constitute target sites for ubiquitylation were mapped; mutation of these Lys residues leads to stabilization of 66K. Altogether, these results demonstrate that TYMV RdRp is a target of the ubiquitin-proteasome system in plant cells and support the idea that proteasomal degradation may constitute yet another fundamental level of regulation of viral replication.

Mutations were introduced by oligonucleotide‐directed mutagenesis into the cDNA of poliovirus type 1 (Mahoney) in the region coding for the first five amino acids (myristoylation signal) of the viral capsid protein precursor P1. The cDNAs were then transcribed in vitro and the properties of the transcripts carrying the mutations studied in vitro by translation in a reticulocyte lysate or in vivo upon transfection of primate cells. Mutation of amino acid residue number 5 (Ser5‐‐‐‐Thr) did not affect the viral phenotype, whereas mutations of residues number 1 (Gly1‐‐‐‐Arg), 2 (Ala2‐‐‐‐Pro) or 5 (Ser5‐‐‐‐Pro) prevented myristoylation of P1 and were lethal. However, delayed production of virus was occasionally observed as the result of reverse mutations, which were found to restore a functional myristoylation signal as well as a wild‐type virus phenotype. Thus, the myristoylation signal of the poliovirus polyprotein can accommodate Ala, Ser, Thr or Leu residues at position 2 and Ser, Thr or Ala residues at position 5. Mutations that altered myristoylation of P1 and affected virus viability did not prevent replication of the viral RNA but severely impeded in vitro processing of P1. This suggests that myristoylation plays a role in poliovirus capsid protein assembly.

A universal molybdenum-containing cofactor (MoCo) is essential for the activity of all human molybdoenzymes, including sulphite oxidase. The free cofactor is highly unstable, and all organisms share a similar biosynthetic pathway. The involved enzymes exhibit homologies, even between bacteria and humans. We have exploited these homologies to isolate a cDNA for the heterodimeric molybdopterin (MPT)-synthase. This enzyme is necessary for the conversion of an unstable precursor into molybdopterin, the organic moiety of MoCo. The corresponding transcript shows a bicistronic structure, encoding the small and large subunits of the MPT-synthase in two different open reading frames (ORFs) that overlap by 77 nucleotides. In various human tissues, only one size of mRNA coinciding with the bicistronic transcript was detected. In vitro translation and mutagenesis experiments demonstrated that each ORF is translated independently, leading to the synthesis of a 10-kDa protein and a 21-kDa protein for the small and large subunits, respectively, and indicated that the 3'-proximal ORF of the bicistronic transcript is translated by leaky scanning.

Turnip yellow mosaic virus (TYMV), a positive-strand RNA virus belonging to the alphavirus-like supergroup, encodes its nonstructural replication proteins as a 206K precursor with domains indicative of methyltransferase (MT), proteinase (PRO), NTPase/helicase (HEL), and polymerase (POL) activities. Subsequent processing of 206K generates a 66K protein encompassing the POL domain and uncharacterized 115K and 85K proteins. Here, we demonstrate that TYMV proteinase mediates an additional cleavage between the PRO and HEL domains of the polyprotein, generating the 115K protein and a 42K protein encompassing the HEL domain that can be detected in plant cells using a specific antiserum. Deletion and substitution mutagenesis experiments and sequence comparisons indicate that the scissile bond is located between residues Ser879 and Gln880. The 85K protein is generated by a host proteinase and is likely to result from nonspecific proteolytic degradation occurring during protein sample extraction or analysis. We also report that TYMV proteinase has the ability to process substrates in trans in vivo. Finally, we examined the processing of the 206K protein containing native, mutated, or shuffled cleavage sites and analyzed the effects of cleavage mutations on viral infectivity and RNA synthesis by performing reverse-genetics experiments. We present evidence that PRO/HEL cleavage is critical for productive virus infection and that the impaired infectivity of PRO/HEL cleavage mutants is due mainly to defective synthesis of positive-strand RNA.

The replication of positive-strand RNA virus genomes is dependent upon the assembly of a replication complex that is an intricate machinery comprising both virus and host components (reviewed in references 1 and 7). Replication complexes are closely associated with intracellular membranes, and many critical interactions among RNA, proteins, and lipids likely take place to allow a successful replication process.Despite the identification and characterization of many viral replication proteins and the purification of a number of positive-strand RNA virus replication complexes that were extensively used in vitro, there is only a limited understanding of the higher-order interactions among these proteins and the ratios in which they are present in viral replication complexes. Understanding how the various proteins interact in these enzyme complexes is essential for unraveling the mechanism of RNA replication and elucidating the three-dimensional structure and function of RNA virus replication complexes. Furthermore, the fine molecular mapping of the interaction sites may constitute an important step toward the identification of the mechanisms that may eventually regulate these interactions or may constitute a way to identify specific targets for new antiviral compounds.To gain insight into the assembly of positive-strand RNA virus replication complexes, we have studied the interactions among the replication proteins of Turnip yellow mosaic virus (TYMV), the type member of the tymovirus group. TYMV is a small spherical plant virus that shares viral replication features with other positive-strand RNA viruses in the alphaviruslike supergroup (17) and has proven useful for investigating fundamental aspects of viral multiplication (3, 60). The TYMV genome is composed of a monopartite, positive-sense RNA of 6,318 nucleotides (Fig. 1A) that directs the expression of two extensively overlapping nonstructural proteins with molecular weights of 69,000 (69K) and 206,000 (206K) (40,61). A third open reading frame (ORF) encodes the 20-kDa coat protein, which is expressed from a subgenomic RNA.The 206K protein is the only viral protein required for TYMV RNA replication (61), and it shows considerable amino acid sequence similarities with nonstructural putative replication proteins of several positive-strand RNA viruses (31). The 206K protein possesses a modular organization, and domains