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1.
Ribosomal initiation from an ACG codon in the Sendai virus P/C mRNA.   总被引:32,自引:7,他引:32       下载免费PDF全文
The Sendai virus P/C mRNA expresses the P and C proteins from alternate reading frames. The C reading frame of this mRNA, however, is responsible for three proteins, C', C and Y, none of which appear to be precursors to each other in vivo. Using site-directed and deletion mutagenesis of the P/C gene cloned in SP6 and in vitro translation of the mRNAs, we show that the 5' most proximal initiation codon of the mRNA is an ACG at position 81, responsible for C' synthesis. The succeeding initiation codons, all ATGs, are responsible for the P protein (position 104), the C protein (position 114) and the Y protein(s) (either positions 183 or 201). Examination of the relative molar amounts of the C', P and C proteins found in vivo suggests that an ACG in an otherwise favorable context is almost as efficient for ribosome initiation as an ATG in a less favored context, but only 10-20% as efficient as an ATG in a more favored context. The judicious choice of increasingly more favorable initiation codons in the P/C gene allows multiple proteins to be made from a single mRNA.  相似文献   

2.
Poliovirus superinfection of Sendai virus-infected cells inhibited the syntheses of the structural P protein and the nonstructural C' and C proteins equally. As the Sendai virus P, C', and C proteins are all translated from the same mRNA by ribosomes which initiate on alternate AUGs and as non-poliovirus protein synthesis is inhibited in poliovirus-infected cells by inactivation of initiation factors responsible for cap group recognition, these results indicate that cap group recognition is important for ribosome initiation on AUGs which are not proximal to the 5' end of the mRNA.  相似文献   

3.
Sendai virus (SeV) P gene encodes a nested set of carboxyl-coterminal proteins (C', C, Y1 and Y2), which are referred to collectively as the C proteins. Characterization of the virus multiplication and cellular responses in HEp-2 cells infected with the recombinant SeV which lacks two (C' and C), three (C', C and Y1) or all the four C proteins revealed that all the recombinant viruses can grow in the cells to various extents, depending, apparently, on the number of species expressing C protein. In reverse proportion to the viral growth ability, these viruses induced apoptosis in the infected cells. These results indicate that Y2 protein has an antiapoptotic activity, and suggest that this activity works in an additive manner with the longer C protein(s) (C' and/or C) of SeV in order to suppress virus-induced apoptosis in the SeV-infected cells. Apparently, the antiapoptotic activity of the C proteins supports virus multiplication in the infected cells.  相似文献   

4.
Parainfluenza virus type 1 (PIV1) and Sendai virus (SEN) are very closely related, but the PIV1 P/C gene does not contain the ACG codon which initiates the SEN C' protein. Nevertheless, a protein corresponding to the PIV1 C' protein was observed both in vivo and in vitro. The initiation site of this protein maps upstream of the PIV1 C protein AUG in a region that does not contain an AUG codon. We have used site-directed mutagenesis to demonstrate that the PIV1 C' protein initiates from a GUG codon, four codons upstream of where the ACG is found in SEN. Remarkably, this GUG appears to initiate in vivo almost as frequently as AUG in the same context. However, whereas GUG permits downstream expression of the P and C proteins, AUG in this context does not. The conservation of an upstream non-AUG initiation codon for C' among PIV1 and SEN suggests that it is important for virus replication, even though some paramyxoviruses express only the C protein and others have no C open reading frame at all.  相似文献   

5.
The polycistronic P/C mRNA of Sendai virus is translated under cell-free conditions into five proteins (P, C', C, Y1, and Y2) from overlapping reading frames. In this study, we showed that in addition to the P, C', and C proteins, Y1 and Y2 were expressed by six different Sendai virus strains in infected cells. The Y proteins exhibited strain-specific variation in their gel mobility which corresponds to the variation seen in the cognate C proteins. While the relative levels of the P, C', and C proteins were consistent among various cell lines, the levels of Y1 and Y2 proteins varied among the cell lines used for viral infection.  相似文献   

6.
Tran EJ  Zhang X  Maxwell ES 《The EMBO journal》2003,22(15):3930-3940
Box C/D ribonucleoprotein (RNP) complexes direct the nucleotide-specific 2'-O-methylation of ribonucleotide sugars in target RNAs. In vitro assembly of an archaeal box C/D sRNP using recombinant core proteins L7, Nop56/58 and fibrillarin has yielded an RNA:protein enzyme that guides methylation from both the terminal box C/D core and internal C'/D' RNP complexes. Reconstitution of sRNP complexes containing only box C/D or C'/D' motifs has demonstrated that the terminal box C/D RNP is the minimal methylation-competent particle. However, efficient ribonucleotide 2'-O-methylation requires that both the box C/D and C'/D' RNPs function within the full-length sRNA molecule. In contrast to the eukaryotic snoRNP complex, where the core proteins are distributed asymmetrically on the box C/D and C'/D' motifs, all three archaeal core proteins bind both motifs symmetrically. This difference in core protein distribution is a result of altered RNA-binding capabilities of the archaeal and eukaryotic core protein homologs. Thus, evolution of the box C/D nucleotide modification complex has resulted in structurally distinct archaeal and eukaryotic RNP particles.  相似文献   

7.
de Breyne S  Stalder R  Curran J 《FEBS letters》2005,579(25):5685-5690
The Sendai virus "C-proteins" (C', C, Y1 and Y2) are a nested set of non-structural proteins. The shorter Y proteins arise in vivo both by de novo translation initiation and by proteolytic processing of C'. In this paper, we demonstrate that C' but not C (differing only by 11 N-terminal amino acid) serves as an efficient substrate for intracellular processing. However, processing can be mimicked in vitro by the addition of endopeptidases. Under conditions of limited proteolysis we observed that in a fraction of the C' protein the Y region exists as a proteinase resistant core. This core was conserved in the C protein. We propose that C' functions as a Pro-protein delivering the Y module to a specific intracellular location.  相似文献   

8.
Expression of proteins from three overlapping genes in a single mRNA species of Sendai virus was modulated in a cell-free rabbit reticulocyte translation system. Hybrid-arrested translation by oligodeoxynucleotides complementary to specific regions of the mRNA that specifies the viral P, C, and C' proteins demonstrated that ribosomes scan the RNA from its 5' end to find initiation codons, and suggested that the secondary structure of the mRNA influences the selection of alternative initiation codons. Translational modulation of P, C, and C' proteins by Mg++ and spermidine indicated that RNA folding is involved in this selection process.  相似文献   

9.
10.
ACG, the initiator codon for a Sendai virus protein   总被引:12,自引:0,他引:12  
Deletion and site-directed point mutants of the polycistronic P/C mRNA of Sendai virus revealed that one of the nonstructural proteins of this virus, the C' protein, initiates from an ACG codon. This ACG codon occurs in an optimum sequence context and precedes the first AUG of the P/C mRNA. The results presented in this communication are consistent with the concept that the ribosomes scan the P/C mRNA to initiate several proteins from its different initiator codons. The arrangement of several weak initiator codons in tandem in an mRNA, i.e. non-AUG in optimum sequence context and AUG in suboptimum sequence context, may represent an alternate means to regulate gene expression in eukaryotes and their viruses.  相似文献   

11.
A fragment of the 16 S ribosomal RNA of Escherichia coli that contains the binding sites for proteins S8 and S15 of the 30 S ribosomal subunit has been isolated and characterized. The RNA fragment, which sediments as 5 S, was partially protected from pancreatic RNAase digestion when S15 alone, or S8 and S15 together, were bound to the 16 S RNA. Purified 5 S RNA was shown to reassociate specifically with protein S15 by analysis of binding stoichiometry. Although interaction between the fragment and protein S8 alone could not be detected, the 5 S RNA selectively bound both S8 and S15 when incubated with an unfractionated mixture of 30-S subunit proteins. Nucleotide sequence analysis demonstrated that the 5 S RNA arises from the middle of the 16 S RNA molecule and encompasses approximately 150 residues from Sections C, C'1 and C'2. Section C consists of a long hairpin loop with an extensively hydrogen-bonded stem and is contiguous with Section C'1. Sections C'1 and C'2, although not contiguous, are highly complementary and it is likely that together they comprise the base-paired stem of an adjacent loop.  相似文献   

12.
The Sendai virus C proteins, C', C, Y1, and Y2, are a nested set of independently initiated carboxy-coterminal proteins translated from a reading frame overlapping the P frame on the P mRNA. The C proteins are extremely versatile and have been shown to counteract the antiviral action of interferons (IFNs), to down-regulate viral RNA synthesis, and to promote virus assembly. Using the stable cell lines expressing the C, Y1, Y2, or truncated C protein, we investigated the region responsible for anti-IFN action and for down-regulating viral RNA synthesis. Truncation from the amino terminus to the middle of the C protein maintained the inhibition of the signal transduction of IFNs, the formation of IFN-stimulated gene factor 3 (ISGF3) complex, the generation of the anti-vesicular stomatitis virus state, and the synthesis of viral RNA, but further truncation resulted in the simultaneous loss of all of these inhibitory activities. A relatively small truncation from the carboxy terminus also abolished all of these inhibitory activities. These data indicated that the activities of the C protein to counteract the antiviral action of IFNs and to down-regulate viral RNA synthesis were not encoded within a region of at least 98 amino acids in its amino-terminal half.  相似文献   

13.
Archaeal box C/D sRNAs guide the methylation of specific nucleotides in archaeal ribosomal and tRNAs. Three Methanocaldococcus jannaschii sRNP core proteins (ribosomal protein L7, Nop56/58, and fibrillarin) bind the box C/D sRNAs to assemble the sRNP complex, and these core proteins are essential for nucleotide methylation. A distinguishing feature of the Nop56/58 core protein is the coiled-coil domain, established by alpha-helices 4 and 5, that facilitates Nop56/58 self-dimerization in vitro. The function of this coiled-coil domain has been assessed for box C/D sRNP assembly, sRNP structure, and sRNP-guided nucleotide methylation by mutating or deleting this protein domain. Protein pull-down experiments demonstrated that Nop56/58 self-dimerization and Nop56/58 dimerization with the core protein fibrillarin are mutually exclusive protein:protein interactions. Disruption of Nop56/58 homodimerization by alteration of specific amino acids or deletion of the entire coiled-coil domain had no obvious effect upon core protein binding and sRNP assembly. Site-directed mutation of the Nop56/58 homodimerization domain also had no apparent effect upon either box C/D RNP- or C'/D' RNP-guided nucleotide modification. However, deletion of this domain disrupted guided methylation from both RNP complexes. Nuclease probing of the sRNP assembled with Nop56/58 proteins mutated in the coiled-coil domain indicated that while functional complexes were assembled, box C/D and C'/D' RNPs were altered in structure. Collectively, these experiments revealed that the self-dimerization of the Nop56/58 coiled-coil domain is not required for assembly of a functional sRNP, but the coiled-coil domain is important for the establishment of wild-type box C/D and C'/D' RNP structure essential for nucleotide methylation.  相似文献   

14.
The P/C mRNA of Sendai virus (SeV) encodes a nested set of accessory proteins, C', C, Y1, and Y2, referred to collectively as C proteins, using the +1 frame relative to the open reading frame of phospho (P) protein and initiation codons at different positions. The C proteins appear to be basically nonstructural proteins as they are found abundantly in infected cells but greatly underrepresented in the virions. We previously created a 4C(-) SeV, which expresses none of the four C proteins, and concluded that the C proteins are categorically nonessential gene products but greatly contribute to viral full replication and infectivity (A. Kurotani et al., Genes Cells 3:111-124, 1998). Here, we further characterized the 4C(-) virus multiplication in cultured cells. The viral protein and mRNA synthesis was enhanced with the mutant virus relative to the parental wild-type (WT) SeV. However, the viral yields were greatly reduced. In addition, the 4C(-) virions appeared to be highly anomalous in size, shape, and sedimentation profile in a sucrose gradient and exhibited the ratios of infectivity to hemagglutination units significantly lower than those of the WT. In the WT infected cells, C proteins appeared to colocalize almost perfectly with the matrix (M) proteins, pretty well with an external envelope glycoprotein (hemagglutinin-neuraminidase [HN]), and very poorly with the internal P protein. In the absence of C proteins, there was a significant delay of the incorporation of M protein and both of the envelope proteins, HN and fusion (F) proteins, into progeny virions. These results strongly suggest that the accessory and basically nonstructural C proteins are critically required in the SeV assembly process. This role of C proteins was further found to be independent of their recently discovered function to counteract the antiviral action of interferon-alpha/beta. SeV C proteins thus appear to be quite versatile.  相似文献   

15.
Box C/D ribonucleoprotein particles guide the 2'-O-ribose methylation of target nucleotides in both archaeal and eukaryotic RNAs. These complexes contain two functional centers, assembled around the C/D and C'/D' motifs in the box C/D RNA. The C/D and C'/D' RNPs of the archaeal snoRNA-like RNP (sRNP) are spatially and functionally coupled. Here, we show that similar coupling also occurs in eukaryotic box C/D snoRNPs. The C/D RNP guided 2'-O-methylation when the C'/D' motif was either mutated or ablated. In contrast, the C'/D' RNP was inactive as an independent complex. Additional experiments demonstrated that the internal C'/D' RNP is spatially coupled to the terminal box C/D complex. Pulldown experiments also indicated that all four core proteins are independently recruited to the box C/D and C'/D' motifs. Therefore, the spatial-functional coupling of box C/D and C'/D' RNPs is an evolutionarily conserved feature of both archaeal and eukaryotic box C/D RNP complexes.  相似文献   

16.
The four Sendai virus C-proteins (C', C, Y1, and Y2) represent an N-terminal nested set of non-structural proteins whose expression modulates both the readout of the viral genome and the host cell response. In particular, they modulate the innate immune response by perturbing the signaling of type 1 interferons. The initiation codons for the four C-proteins have been mapped in vitro, and it has been proposed that the Y proteins are initiated by ribosomal shunting. A number of mutations were reported that significantly enhanced Y expression, and this was attributed to increased shunt-mediated initiation. However, we demonstrate that this arises due to enhanced proteolytic processing of C', an event that requires its very N terminus. Curiously, although Y expression in vitro is mediated almost exclusively by initiation, Y proteins in vivo can arise both by translation initiation and processing of the C' protein. To our knowledge this is the first example of two apparently independent pathways leading to the expression of the same polypeptide chain. This dual pathway explains several features of Y expression.  相似文献   

17.
Vesicular stomatitis virus (VSV) has served for several decades as the prototype rhabdovirus and a model RNA virus. Extensive studies upheld the original view of VSV genetics with simply five genes (N, P, M, G, and L), each encoding a single unique protein. We now report the first unambiguous demonstration of the existence of an additional unique protein encoded in an overlapping frame within the virus P gene. Experiments using antipeptide sera specific for the predicted second open reading frame have demonstrated the synthesis of two N-terminally nested forms of the protein in virus-infected cells. The major form is 55 amino acids in length, whereas the minor form has 10 additional N-terminal amino acids. Ribosome initiation of synthesis of these proteins appears to occur at AUG codons, 68 and 41 bases, respectively, downstream of the P protein AUG initiation codon. The proteins are found in the cytoplasm of the infected cell but are undetectable in purified virions, consistent with their being nonstructural proteins. Both the major and minor forms of the protein are highly basic and arginine rich, reminiscent of the C and C' proteins encoded in overlapping frame close to the 5' terminus of the P mRNA of several paramyxoviruses. The potential to encode small, highly basic proteins within the P mRNA 5' terminus is highly conserved among the vesiculoviruses.  相似文献   

18.
A Mn2+-dependent protein phosphatase 2A which is composed of a 34 kDa catalytic C' subunit and a 63 kDa regulatory A' subunit, was purified from human erythrocyte cytosol. C' and A' produced V8- and papain-peptide maps identical to those of the 34 kDa catalytic C and the 63 kDa regulatory A subunits of the Mn2+-independent conventional protein phosphatase in human erythrocyte cytosol, respectively. Reconstitution of C'A and CA' revealed that the metal dependency resided in C' and not in A'. In CA, 0.87 +/- 0.12 mol zinc and 0.35 +/- 0.18 mol iron per mol enzyme were detected by atomic absorption spectrophotometry, but manganese, magnesium and cobalt were not detected. None of these metals was detected in C'A'. Pre-incubation of C' with ZnCl2 and FeCl2, but not FeCl3, synergistically stimulated the Mn2+-independent protein phosphatase activity. The protein phosphatase activity of C was unaffected by the same zinc and/or iron treatment. These results suggest that C is a Zn2+- and Fe2+-metalloenzyme and that C' is the apoenzyme.  相似文献   

19.
TNfn3, the third fibronectin type III domain of human tenascin, is an immunoglobulin-like protein that is a good model for experimental and theoretical analyses of Greek key folding. The third fibronectin type III domain of human tenascin folds and unfolds in a two-state fashion over a range of temperature and pH values, and in the presence of stabilising salts. Here, we present a high resolution protein engineering analysis of the single rate determining transition state. The 48 mutations report on the contribution of side-chains at 32 sites in the core and loop regions. Three areas in the protein exhibit high Phi-values, indicating that they are partially structured in the transition state. First, a common-core ring of four positions in the central strands B, C, E and F, that are in close contact, form a nucleus of tertiary interactions. The two other regions that appear well-formed are the C' region and the E-F loop. The Phi-values gradually decrease away from these regions such that the very ends of the two terminal strands A and G, have Phi-values of zero. We propose a model for the folding of immunoglobulin-like proteins in which the common-core "ring" forms the nucleus for folding, whilst the C' and E-F regions are constrained by topology to pack early. Folding characteristics of a group of structurally related proteins appear to support this model.  相似文献   

20.
Spitznagel, John K. (University of North Carolina School of Medicine, Chapel Hill). Normal serum cytotoxicity for P(32)-labeled smooth Enterobacteriaceae. III. Isolation of a gammaG normal antibody and characterization of other serum factors causing P(32) loss. J. Bacteriol. 91:401-408. 1966.-Gram-negative bacteria lost metabolically incorporated P(32) when suspended in serum only if the serum contained heat-labile in addition to heat-stable factors. Gram-positive bacteria labeled with P(32) and included for comparison lost P(32) in heat-inactivated as well as in fresh normal serum. Further investigation of gram-negative bacteria showed that a smooth Escherichia coli (O117:H27) lost P(32) only if suspended in serum containing complement fractions C'1, C'2, C'3, and C'4 "normal" antibody and lysozyme. The normal antibody was recovered by absorption on and subsequent elution from E. coli O117:H27 cell walls. Immunoelectrophoresis showed that it was a gammaG-globulin. Its P(32)-releasing activity was destroyed by 2-mercaptoethanol. Lysozyme was found to potentiate the P(32)-releasing action of normal antibody plus complement. Evidence was obtained suggesting that beta(1C) globulin was the component absorbed to zymosan during serum absorption at 15 C. Reduction of the beta(1C) level evidently upsets an important balance that exists in normal serum among complement, antibody, and lysozyme. This balance is essential for maximal P(32) release from labeled bacteria, or possibly for a maximal antibacterial effect from normal serum. The possible relationships of bactericidal, bacteriolytic, and opsonic action of normal serum are discussed.  相似文献   

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