Presence Of Sparsomycin Research Articles

Rapid Propagation of Low-Fitness Drug-Resistant Mutants of Human Immunodeficiency Virus Type 1 by a Streptococcal Metabolite Sparsomycin

Here we report that sparsomycin, a streptococcal metabolite, enhances the replication of HIV-1 in multiple human T cell lines at a concentration of 400 nM. In addition to wild-type HIV-1, sparsomycin also accelerated the replication of low-fitness, drug-resistant mutants carrying either D30N or L90M within HIV-1 protease, which are frequently found mutations in HIV-1-infected patients on highly active antiretroviral therapy (HAART). Of particular interest was that replication enhancement appeared profound when HIV-1 such as the L90M-carrying mutant displayed relatively slower replication kinetics. The presence of sparsomycin did not immediately select the fast-replicating HIV-1 mutants in culture. In addition, sparsomycin did not alter the 50% inhibitory concentration (IC50) of antiretroviral drugs directed against HIV-1 including nucleoside reverse transcriptase inhibitors (lamivudine and stavudine), non-nucleoside reverse transcriptase inhibitor (nevirapine) and protease inhibitors (nelfinavir, amprenavir and indinavir). The IC50s of both zidovudine and lopinavir against multidrug resistant HIV-1 in the presence of sparsomycin were similar to those in the absence of sparsomycin. The frameshift reporter assay and Western blot analysis revealed that the replication-boosting effect was partly due to the sparsomycin's ability to increase the -1 frameshift efficiency required to produce the Gag-Pol transcript. In conclusion, the use of sparsomycin should be able to facilitate the drug resistance profiling of the clinical isolates and the study on the low-fitness viruses.

Ribosomal Proteins Cross-Linked to the Initiator AUG Codon of a mRNA in the Translation Initiation Complex by UV-Irradiation

Eukaryotic ribosomal proteins constituting the binding site for the initiator codon AUG on the ribosome at the translation initiation step were investigated by UV-induced cross-linking between protein and mRNA. The 80S-initiation complex was formed in a rabbit reticulocyte cell-free system in the presence of sparsomycin with radiolabeled Omega-fragment as a template, which was a 73-base 5'-leader sequence of tobacco mosaic virus RNA having AUG at the extreme 3'-terminal end and extended with 32pCp. Two radioactive peaks were sedimented by sucrose gradient centrifugation, one being the 80S initiation complex formed at the 3'-terminal AUG codon, and the other presumably a "disome" with an additional 80S ribosome bound at an upstream AUU codon, formed when Omega-fragment was incubated with sparsomycin [Filipowicz and Henni (1979) Proc. Natl. Acad. Sci. USA 76, 3111-3115]. Cross-links between ribosomal proteins and the radiolabeled Omega-fragment were induced in situ by UV-irradiation at 254 nm. After extensive nuclease digestion of the complexes, ribosomal proteins were separated by two-dimensional gel electrophoresis. Autoradiography identified the proteins S7, S10, S25, S29, and L5 of the 80S initiation complex and S7, S25, S29 and L5 of that in the disome as 32P-labeled proteins. Together with the results of cross-linking experiments of other investigators and recently solved crystal structures of prokaryotic ribosomes, the spatial arrangement of eukaryotic ribosomal proteins at the AUG-binding domain is discussed.

Sites of interaction of the CCA end of peptidyl-tRNA with 23S rRNA.

Oligonucleotide fragments derived from the 3' CCA terminus of acylated tRNA, such as CACCA-(AcPhe), UACCA-(AcLeu), and CAACCA-(fMet), bind specifically to ribosomes in the presence of sparsomycin and methanol [Monro, R. E., Celma, M. L. & Vazquez, D. (1969) Nature (London) 222, 356-358]. All three oligonucleotides protect a characteristic set of bases in 23S rRNA from chemical probes: G2252, G2253, A2439, A2451, U2506, and U2585. A2602 shows enhanced reactivity. These account for most of the same bases that are protected when peptidyl-tRNA analogues such as AcPhe-tRNA are bound to the ribosomal P site, and correspond precisely to those bases whose protection is abolished by removal of the 3'-CA end of tRNA. We conclude that most of the observed interactions between tRNA and 23S rRNA in the 50S ribosomal P site involve the conserved CCA terminus of tRNA. Sparsomycin may inhibit protein synthesis by stabilizing interaction between the peptidyl-CCA and the 23S P site, preventing formation of the intermediate A/P hybrid state.

Complete uncoating of the 5' leader sequence of tobacco mosaic virus RNA occurs rapidly and is required to initiate cotranslational virus disassembly in vitro

Destabilizing events required for subsequent cotranslational disassembly of tobacco mosaic virus (TMV) particles in vitro were studied. Brief treatment of U-32P-labelled TMV (strain vulgare or U2) with 1% SDS exposed only 2.5% of the RNA (160 5' nucleotides) in a susceptible subpopulation of virions. Limited uncoating occurred almost immediately and appeared to be synchronous because the amount of 5' oligonucleotide marker (omega) recovered remained constant throughout a 15 min period in SDS. Additional RNase T1-sensitive oligonucleotides were exposed only after 1 to 2 min in SDS. Coat protein (CP) subunits released from virions 'destabilized' by ultracentrifugation at between pH 7.2 and 9.2 were quantified using L-[35S]methionine-labelled particles of TMV strain U2. CP recovery and virus particle translation results were consistent with increasing numbers of virions uncoating for approximately 200 nucleotides. In the presence of sparsomycin (SPN), the TMV strain vulgare 5' leader and the first AUG codon can bind two 80S ribosomes. Electron microscopy of pH 7.5-treated TMV particles incubated in SPN-treated wheatgerm extract or rabbit reticulocyte lysate, showed that approximately 10% of virions complexed with one ribosome and approximately 10% with two bound ribosomes, confirming that omega at least had been uncoated. Nucleocapsids in these complexes were shorter than untreated TMV by 9 to 10 nm (i.e. equivalent to 192 to 217 nucleotides exposed). The template activities of virions pretreated at pH 7.2 to 9.2 were destroyed by RNase H when short cDNAs were hybridized to sequences at, or immediately 3' to, the first AUG codon. We propose that the complete 5' leader of TMV RNA interacts weakly with CP subunits and that this micro-instability is due to the absence of G residues and is essential for initiation of cotranslational virus disassembly.

Effect of cupric ions on the initiation protein synthesis rate in the human endometrium

The effect of cupric ions on the initiation protein synthesis rate of the human endometrium was studied. Addition of copper to the complete ribosomal system decreased the binding of [ 3H]Met-tRNA(i) to the isolated ribosomes with a plateau at about 70% inhibition with concentrations higher than 150 μM. The initiation activity was GTP-dependent with a maximum at 2 mM. This activity was very rapid, requiring 5 min to complete the reaction. Incubation of isolated initiation factors with copper (300 μM) inhibited the formation of the ternary complex. When the complete system was reconstituted with salt-washed ribosomes after ternary complex formation, no significant change on the inhibition pattern was observed. Addition of initiation factors to 5-min preincubated salt-washed ribosomes with 300 μM copper, after the elimination of excess copper, induced only a 12% decrease on Met-tRNA(i) binding. This effect was not modified by the presence of Sparsomycin, an elongation inhibitor. It was concluded that copper interferes with the initiation process, probably at the ternary complex formation level.

In Vitro Synthesis, Phosphorylation, and Localization on 48 S Initiation Complexes of Human Protein Synthesis Initiation Factor 4E

Complementary DNA for human eukaryotic initiation factor 4E (eIF-4E) was transcribed in vitro and the transcripts used to direct protein synthesis in a cell-free reticulocyte translation system. The predominant translation product was 25 kDa, was bound to a m7GTP-Sepharose affinity column, and was specifically eluted with m7GTP. Both phosphorylated (P) and unphosphorylated (U) forms of eIF-4E were synthesized, and the P/U ratio increased as a function of incubation time in the reticulocyte lysate system. Both forms were quantitatively retained on m7GTP-Sepharose. When translation reactions were resolved on sucrose density gradients, the 35S-labeled eIF-4E sedimented predominantly at 3-4 S. However, in the presence of edeine or guanylyl imidodiphosphate, both of which cause accumulation of 48 S initiation complexes, eIF-4E was detected in the 48 S region. In the presence of sparsomycin, used to accumulate 80 S initiation complexes, no eIF-4E was observed in the 80 S region. No change in the eIF-4E distribution was caused by m7GTP. These results are consistent with a model whereby eIF-4E is transferred to the 43 S initiation complex together with mRNA and is released from the initiation complex when the 60 S ribosomal subunit joins.

Studies on the mechanism of translational enhancement by the 5'-leader sequence of tobacco mosaic virus RNA.

Translation of foreign mRNAs is enhanced by a cis-acting derivative (omega') of the 5'-leader sequence (omega) of tobacco mosaic virus RNA (vulgare strain). To explain this effect we have conducted several experiments in vitro. 1. The presence of various 5'-terminal sequences, including omega', did not significantly increase the half-lives of chloramphenicol acetyltransferase (CAT) or neomycin phosphotransferase (NPTII) mRNAs in wheat-germ extract. Also, a long leader sequence, unrelated to omega', did not enhance expression of NPTII mRNA in vitro. 2. The ability of several leader sequences, including omega', to form multiple initiation complexes with 80S (wheat germ) ribosomes was examined using CAT or NPTII mRNAs incubated in the presence of sparsomycin. Formation of disome complexes was unrelated to the capacity of a 5'-leader sequence to enhance translation. 3. Expression of CAT mRNA in both wheat germ extract and messenger-dependent rabbit reticulocyte lysate was less susceptible to inhibition by increasing salt concentration when a 5'-proximal omega' sequence was present. This effect was less marked when the CAT mRNA was capped. Conversely at high salt concentrations, capping was less stimulatory for mRNA with a 5'-proximal omega' sequence. These data suggest that omega' and the cap enhance translation, at least in part, by a similar mechanism. We propose that both features reduce RNA secondary structure, thereby rendering the 5' terminus more accessible to scanning by 40S ribosomal subunits and/or interaction with associated initiation factors. This conclusion was supported by computer-based secondary-structure analyses of our SP6 RNA polymerase transcript sequences. The ability of 5' leader sequences from brome mosaic virus RNA 3, alfalfa mosaic virus RNA 4, and the genomic RNAs of turnip yellow mosaic virus, Rous sarcoma virus or tobacco mosaic virus (tomato strain) to enhance mRNA translation in eukaryotic systems may also be correlated with their respective secondary structures. A different mechanism probably accounts for the omega'-dependent enhancement of mRNA expression in Escherichia coli or in E. coli cell-free systems.

Higher order structures of the 5'-proximal region decrease the efficiency of translation of the porcine pro-opiomelanocortin mRNA.

The SP6 polymerase/promoter system was used to synthesize porcine pro-opiomelanocortin mRNAs with nucleotide sequence deletions in the 5'- as well as 3'-untranslated and coding regions. The translational efficiency of the mutant mRNAs was evaluated by cell-free translation or by monitoring the rate and extent of ribosome binding in the presence of sparsomycin. The results of these experiments indicate that specific nucleotide sequences in the 5'-untranslated and coding regions of the pro-opiomelanocortin mRNA decrease its rate of translation. Structure mapping of the mRNA with double-strand and single-strand specific nucleases suggests that these sequences can form stable secondary structures.

Attachment of the 5'-terminal portion of globin mRNAs to 5S-RNA X L5-protein in the 80S initiation complex.

An 80S initiation complex was formed by incubating a heterologous cell-free system with 125I-labeled globin mRNAs in the presence of sparsomycin. The 80S initiation complex was then digested with micrococcal nuclease. The ribosomal 5S-RNA X L5-protein (5S RNP) fraction, released by EDTA treatment, contained 125I-labeled mRNA fragments. The attachment of labeled mRNA fragments to 5S RNP was shown by (a) CsCl isopycnic centrifugation, (b) recentrifugation through a sucrose density gradient and (c) acrylamide gel electrophoresis of 5S RNP purified by (b). Labeled fragments were released from 5S RNP by treatment with sodium dodecyl sulfate or pronase, indicating the participation of protein L5 in the attachment. The attached mRNA fragments were 23-25 nucleotides in length. Hybridization experiments, using restriction fragments of cDNA for rabbit beta globin mRNA, showed that the attached mRNA fragments were derived from the 5' portion of globin mRNAs. The attachment of 125I-labeled mRNA fragments to 5S RNP was also observed in the 80S initiation complex formed by incubation of reticulocyte lysate with 125I-labeled globin mRNA, but not in labeled polysomal fractions. These findings may indicate that 5S RNP interacted with the 5' portion of globin mRNA, containing the translation initiation codon of globin mRNA in the 80S initiation complex. The biochemical significance of these results is discussed.

Inhibition of globin chain initiation in reticulocyte lysates by pactamycin: Accumulation of methionyl-valine

In a globin synthesizing system containing reticulocyte lysate and [ 35S] met-tRNA f, low levels of pactamycin cause an accumulation of radioactivity on the monosomes and small oligosomes concomitant with the breakdown of polysomes. About 50% of the ribosome-bound radioactivity corresponds to methionyl-valine, the initial dipeptide of globin chains, with negligible amounts of tri- or other oligopeptides. This suggests that the site of its action is after the formation of the first peptide bond. The ribosome-bound radioactivity in the presence of sparsomycin, an inhibitor of chain elongation, is in di-, tri- and oligopeptides. Sparsomycin levels (10 −5M) that cause almost complete inhibition of polypeptide synthesis have only a small inhibitory effect on the pactamycin-induced accumulation of methionyl-valine. Fusidic acid and chlortetracycline do not cause accumulation of any significant amounts of methionyl-valine.

Enhancement of the phenylalanyl-oligonucleotide binding to the peptidyl recognition center of ribosomal peptidyltransferase and inhibition of the chloramphenicol binding to ribosomes

The binding of C-A-C-C-A(Phe) to ribosomes was critically affected by the concentration of ethanol and C-A-C-C-A(Phe) in the reaction mixture. In the presence of sparsomycin, the binding was inhibited at a low ethanol concentration, whereas it was stimulated at a moderately high ethanol concentration. At a moderately high concentration of ethanol, plots of the initial velocity of C-A-C-C-A(Phe) binding to ribosomes against C-A-C-C-A(Phe) concentration exhibited a sigmoidal saturation curve; while at low ethanol concentration, it gave a hyperbolic saturation curve. C-A-C-C-A(Phe) could not react with puromycin if a low level of C-A-C-C-A(Phe) and a low concentration of ethanol were included in the reaction mixture, while it did react with puromycin to form phenylalanyl-puromycin when a high level of C-A-C-C-A(Phe) and a high concentration of ethanol were used. In the presence of a moderately high concentration of ethanol, the binding of chloramphenicol to ribosomes was markedly inhibited by C-A-C-C-A(Phe) or C-A-C-C-A(Ac-Phe), while sparsomycin enhanced the inhibition. In the absence of ethanol, the phenylalanyl-oligonucleotides slightly affected the chloramphenicol binding to ribosomes. On the basis of these findings, it was assumed that C-A-C-C-A(Phe) could bind to the peptidyl recognition center as well as the aminoacyl recognition center of ribosomes, and that chloramphenicol could bind to the ribosomes at or very close to the peptidyl recognition center of the peptidyl transferase.

Amino acylaminonucleoside inhibitors of protein synthesis: II. Effect on oligophenylalanine formation

1. 1. The inhibition of the poly U-directed incorporation of phenylalanyl residues from l-phenylalanyl-tRNA into peptide chains by a number of antibiotics was studied in a ribosomal system from Escherichia coli. The inhibitors were amicetin, blasticidin S, chloramphenicol, gougerotin and sparsomycin. The products of incorporation were examined by column chromatography on benzoylated diethylaminoethyl cellulose. The fractions obtained were: phenylalanylphenylalanine ( Phe) 2 oligophenylalanine ( oligo Phe) and polyphenylalanine ( poly Phe). The incorporation was carried out at 10 mM Mg 2+ in the presence of various amounts of transfer factors. The rate of incorporation of phenylalanyl residues into peptide linkage as well as the distribution of peptide chain lengths varied with the concentration of these factors. 2. 2. In the presence of amicetin, blasticidin S, gougerotin or chloramphenicol, oligopeptides ( Phe) 2 and oligo( Phe) accumulated and constituted the major product of phenylalanine incorporation. Despite initial interference with ( Phe) 2, oligo Phe and poly Phe formation, the total number of peptide bonds (( Phe) 2, oligo Phe and poly Phe formed after 8 min in the presence of these inhibitors was equal to, or exceeded the number of peptide bonds obtained in their absence. At the same time, the amount of poly Phe formed in the presence of the inhibitors was consistently lower than in the control. The total phenylalanine incorporation into peptide bonds in the absence of inhibitors proceeded linearly for the entire time period examined (32 min). Oligopeptides were also accumulated in the presence of sparsomycin but total peptide bond formation was always less than in the control. Tetracycline did not cause the accumulation of oligopeptides. 3. 3. The results obtained with amicetin, blasticidin S, gougerotin or chloramphenicol cannot be explained solely on the basis of a constant interference with peptide bond formation by these antibiotics. Alternative explanations are considered.