Composition, arrangement and cleavage of the mouse mammary tumor virus polyprotein precursor Pr77gag and p110gag.

The amino acid sequence relationship between the nonglycosylated structural proteins of murine mammary tumor virus and the polyproteins from infected cells immunoprecipitated with an anti-p27 serum were examined using two-dimensional tryptic peptide mapping procedures. The proteins were labeled with ¹⁴C-lysine and ¹⁴C-arginine so that all but one of the tryptic peptides released from a protein could be detected. Previous studies have shown that immunoprecipitation of mammary tumor cells with anti-p27 serum results in the isolation of seven proteins in the molecular weight range of 34,000–160,000 daltons; and that cell-free translation using viral genomic RNA yields three p27-related proteins of 160,000, 110,000 and 77,000 daltons, similar to the three high molecular weight proteins detected in vivo. The proteins of lower molecular weight were thought to be cleavage intermediates of Pr77^gag. As judged from the peptide maps, Pr77^gag contained the complete sequences of the four major internal proteins of the virion (p27, pp21, p14 and p10) and possibly a fifth highly basic protein (p8) also found in virions. The putative cleavage intermediates, as expected, lacked some tryptic peptides that could be assigned to one or more of the major virion proteins and thus allow a scheme for the cleavage events to be constructed. p110^gag contained all the tryptic peptides found in Pr77^gag, plus some additional peptides. A minor virion protein p30 was found to include the peptides of p14 as well as some of the additional peptides present in p110^gag, suggesting a precursor-product relationship between the pr110^gag and p30. The data obtained from these studies lead us to propose that there are three protein precursors which include, at least in part, the gag gene region of the virion—p160 (potentially a gag/pol precursor), p110^gag and Pr77^gag—and that the arrangement of the virion proteins within the gag gene (pr77^gag) is p10-pp21-p27-p14.     

Suppression of Murine Retrovirus Polypeptide Termination: Effect of Amber Suppressor tRNA on the Cell-Free Translation of Rauscher Murine Leukemia Virus, Moloney Murine Leukemia Virus, and Moloney Murine Sarcoma Virus 124 RNA

The effect of suppressor tRNA's on the cell-free translation of several leukemia and sarcoma virus RNAs was examined. Yeast amber suppressor tRNA (amber tRNA) enhanced the synthesis of the Rauscher murine leukemia virus and clone 1 Moloney murine leukemia virus Pr200^gag-pol polypeptides by 10- to 45-fold, but at the same time depressed the synthesis of Rauscher murine leukemia virus Pr65^gag and Moloney murine leukemia virus Pr63^gag. Under suppressor-minus conditions, Moloney murine leukemia virus Pr70^gag was present as a closely spaced doublet. Amber tRNA stimulated the synthesis of the “upper” Moloney murine leukemia virus Pr70^gag polypeptide. Yeast ochre suppressor tRNA appeared to be ineffective. Quantitative analyses of the kinetics of viral precursor polypeptide accumulation in the presence of amber tRNA showed that during linear protein synthesis, the increase in accumulated Moloney murine leukemia virus Pr200^gag-pol coincided closely with the molar loss of Pr63^gag. Enhancement of Pr200^gag-pol and Pr70^gag by amber tRNA persisted in the presence of pactamycin, a drug which blocks the initiation of protein synthesis, thus arguing for the addition of amino acids to the C terminus of Pr63^gag as the mechanism behind the amber tRNA effect. Moloney murine sarcoma virus 124 30S RNA was translated into four major polypeptides, Pr63^gag, P42, P38, and P23. In the presence of amber tRNA, a new polypeptide, Pr67^gag, appeared, whereas Pr63^gag synthesis was decreased. Quantitative estimates indicated that for every 1 mol of Pr67^gag which appeared, 1 mol of Pr63^gag was lost.     

P85: a gag-mos polyprotein encoded by ts110 Moloney murine sarcoma virus

Antibody to a synthetic peptide (anti-C3 serum) with the predicted sequence of the C terminus of the Moloney murine sarcoma virus (strain 124) v-mos gene was used in immunoprecipitation experiments with cytoplasmic extracts of a clone of NRK cells infected with ts110 Moloney murine sarcoma virus, termed 6m2 cells. ts110 Moloney murine sarcoma virus codes for two viral proteins of 85,000 and 58,000 M_r, termed P85 and P58, respectively, in nonproducer 6m2 cells maintained at 33°C. Anti-C3 serum specifically recognized [³H]leucine-labeled P85, but not P58, from infected cells maintained at 33°C, whereas antiserum prepared against murine leukemia virus p12 recognized both proteins. Normal serum and anti-C3 serum pretreated with excess C3 peptide did not precipitate P85. Immunoprecipitation experiments after metabolic labeling of 6m2 cells with ³²P_i showed that P85 is phosphorylated. Both anti-C3 and anti-p12 sera specifically detected ³²P-labeled P85. Cell-free translation of ts110 murine sarcoma virus/murine lukemia virus RNA produces P85, P58, and helper virus protein Pr63^gag. Anti-C3 serum specifically precipitated P85 but neither P58 nor Pr63^gag. We conclude from these studies that P85 is a product of both the gag and mos genes of ts110 murine sarcoma virus, and, therefore, it is referred to as P85^gag-mos. We have not detected any other v-mos gene product in ts110-infected cells.     

High-level expression of the HIV-1 Pr55gag polyprotein in transgenic tobacco chloroplasts

Plants have been recognized as a promising production platform for recombinant pharmaceutical proteins. The human immunodeficiency virus Gag (Pr55^gag) structural polyprotein precursor is a prime candidate for developing a HIV-1 vaccine, but, so far, has been expressed at very low level in plants. The aim of this study was to investigate factors potentially involved in Pr55^gag expression and increase protein yield in plant cells. In transient expression experiments in various subcellular compartments, the native Pr55^gag sequence could be expressed only in the chloroplast. Experiments with truncated subunits suggested a negative role of the 5′-end on the expression of the full gene in the cytosol. Stable transgenic plants were produced in tobacco by Agrobacterium-mediated nuclear transformation with protein targeted to plastids, and biolistic-mediated plastid transformation. Compared to the nuclear genome, the integration and expression of the gag transgene in the plastome resulted in significantly higher protein accumulation levels (up to 7–8% TSP, equivalent to 312–363 mg/kg FW). In transplastomic plants, a 25-fold higher protein accumulation was obtained by translationally fusing the Pr55^gag polyprotein to the N-terminus of the plastid photosynthetic RbcL protein. In chloroplasts, the Pr55^gag polyprotein was processed in a pattern similar to that achieved by the viral protease, the processing being more extended in older leaves of mature plants. The Gag proteins produced in transgenic plastids were able to assemble into particles resembling VLPs produced in baculovirus/insect cells and E. coli systems. These results indicate that plastid transformation is a promising tool for HIV antigen manufacturing in plant cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. IGV publication no. 330     

Species-specific effects of elevated CO2 on resource allocation in Plantago maritima and Armeria maritima

We investigated the effects of increased atmospheric CO₂ on the biomass, photosynthesis, protein and phenolic concentrations and content of Plantago maritima and Armeria maritima. This enabled us to test the protein competition model (PCM) for predicting C allocation to phenolics. Three contrasting responses to elevated CO₂ (600 μmol CO₂ mol⁻¹) between the two study species were observed. (1) In P. maritima, plant biomass increased and the maximum carboxylation rate of Rubisco (V_c,max) was decreased. However, in A. maritima, shoot biomass decreased and the V_c,max of Rubisco was unchanged. (2) The total phenolic content increased in P. maritima but decreased in A. maritima. (3) Protein concentrations and content decreased in P. maritima and root protein concentrations and content increased in A. maritima. We conclude that C and N allocation to phenolics and proteins is species- and organ-specific and the PCM predictions were correct when phenolics and proteins were expressed on a per plant content basis.     

Insertion of newly synthesized proteins into the outer membrane of Escherichia coli

The insertion of newly synthesized proteins into the outer membrane of Escherichia coli has been examined. The results show that there is no precursor pool of outer membrane proteins in the cytoplasmic membrane because first, the incorporation of a [³⁵S]methionine pulse into outer membrane proteins completely parallels its incorporation into cytoplasmic membrane proteins, and second, under optimal isolation conditions, no outer membrane proteins are found in the cytoplasmic membrane, even when the membranes are analysed after being labeled for only 15 s.The [³⁵S]methionine present in the outer membrane after a pulse of 15 s was found in protein fragments of varying sizes rather than in specific outer membrane proteins. This label could however be chased into specific proteins within 30–120 s, depending on the size of the protein, indicating that although unfinished protein fragments were present in the outer membrane, they were completed by subsequent chain elongation.Thus, outer membrane proteins are inserted into the outer membrane while still attached to ribosomes. Since ribosomes which are linked to the cell envelope by nascent polypeptide chains are stationary, the mRNA which is being translated by these ribosomes moves along the inner cell surface.     

Proline 146 is critical to the structure, function and targeting of sod2, the Na/H exchanger of Schizosaccharomyces pombe

Sod2 is the Na⁺/H⁺ exchanger of the fission yeast Schizosaccharomyces pombe that is principally responsible for salt tolerance. We examined the role of nine polar, membrane associated amino acids in the ability of the protein to confer salt tolerance in S. pombe. Wild type sod2 protein with a C-terminal GFP tag effectively rescued salt tolerance in S. pombe with deleted endogenous sod2. Sod2 protein with the mutations P163A, P183A, D298N, D389N, E390Q, E392Q and E397Q also conveyed salt tolerance as effectively as the wild type sod2 protein. In contrast, the mutation P146A resulted in a protein that did not convey salt tolerance nearly as effectively as the wild type and did not extrude Na⁺ as well as the wild type. Mutation of Pro¹⁴⁶ to Ser, Asp or Lys had an intermediate effect. Mutation of Thr¹⁴² to Ser resulted in a slightly defective protein. Western blot analysis showed that all mutant proteins were expressed at similar levels as wild type sod2 protein. Examination of the localization of the proteins showed that wild type and most sod2 mutants were present in the plasma membrane while the P146A mutant had an intracellular localization. Limited tryptic digestion suggested that the P146A sod2 protein had a change in conformation in comparison to the wild type protein. The results suggest that Pro¹⁴⁶ is an amino acid critical to sod2 structure, function and localization.     

Protein phosphorylation regulates in vitro spinach chloroplast petD mRNA 3′-untranslated region stability,processing, and degradation

RNA-binding proteins (RNPs) participate in diverse processes of mRNA metabolism, and phosphorylation changes their binding properties. In spinach chloroplasts, 24RNP and 28RNP are associated with polynucleotide posphorylase forming a complex on charge of pre-mRNA 3′-end maturation. Here, we tested the hypothesis that the phosphorylation status of 24RNP and 28RNP, present in a spinach chloroplast mRNA 3′-UTR processing extract (CPE), controls the transition between petD precursor stabilization, 3′-UTR processing, and RNA degradation in vitro. The CPE processed or stabilized petD precursor depending on the ATP concentration present in an in vitro 3′-UTR processing (IVP) assay. These effects were also observed when ATP was pre-incubated and removed before the IVP assay. Moreover, a dephosphorylated (DP)-CPE degraded petD precursor and recovered 3′-UTR processing or stabilization activities in an ATP concentration dependent manner. To determine the role 24/28RNP plays in regulating these processes a 24/28RNP-depleted (Δ24/28)CPE was generated. The Δ24/28CPE degraded the petD precursor, but when it was reconstituted with recombinant non-phosphorylated (NP)-24RNP or NP-28RNP, the precursor was stabilized, whereas when Δ24/28CPE was reconstituted with phosphorylated (P)-24RNP or P-28RNP, it recovered 3′-UTR processing, indicating that 24RNP or 28RNP is needed to stabilize the precursor, have a redundant role, and their phosphorylation status regulates the transition between precursor stabilization and 3′-UTR processing. A DP-Δ24/28CPE reconstituted or not with NP-24/28RNP degraded petD precursor. Pre-incubation of DP-Δ24/28CPE with NP-24/28RNP plus 0.03 mM ATP recovered 3′-UTR processing activity, and its reconstitution with P-24/28RNP stabilized the precursor. However, pre-incubation of DP-Δ24/28CPE with 0.03 mM ATP, and further reconstitution with NP-24/28RNP or P-24/28RNP produced precursor stability instead of RNA degradation, and RNA processing instead of precursor stability, respectively. Moreover, in vitro phosphorylation of CPE showed that 24RNP, 28RNP, and other proteins may be phosphorylated. Altogether, these results reveal that phosphorylation of 24RNP, 28RNP, and other unidentified CPE proteins mediates the in vitro interplay between petD precursor stability, 3′-UTR processing, and degradation, and support the idea that protein phosphorylation plays an important role in regulating mRNA metabolism in chloroplast.